The formation of synapses and their stabilization occur principally during nervous system development. It is the intricate specificity of synapses and associated neural networks which form the scaffolding upon which higher order cognitive and behavioral functions are acquired. Disruptions in normal synapse formation may underlie certain forms of learning disorders and mental illness. One synaptic component of critical importance is the voltage-dependent N-type calcium channel (N-type VDCC). The entry of calcium through N-type VDCC in mature neurons promotes the release of neurotransmitters via calcium-dependent exocytosis of synaptic vesicles. Several lines of evidence suggest that N-type VDCC is also involved in the signalling mechanism that promotes growth cone extension and neurite elongation in developing neurons. The structure of N-type VDCC in developing neurons, the assembly of complexes throughout ontogeny, and the role of N-type VDCC in formation of mature synapses is currently unexplored. The goal of this proposal is to investigate the subunit composition of the N-type VDCC expressed throughout rat brain ontogeny. The long term objective of our studies is to understand the molecular and cellular mechanisms underlying the expression and assembly of N-type VDCC subunits in neuronal differentiation and the contribution of N-type VDCC function to synapse formation. Calcium channels present in the plasma membrane of mature neurons are multimeric complexes composed of a channel-forming alpha1 subunit, a glycosylated alpha2 subunit, and a beta subunit. There are four isoforms of beta subunit identified by molecular cloning. Our current hypothesis is that alterations in the subunit composition of the N-type VDCC subunit, specifically in the association of beta and alpha1 subunits, occur during neuronal maturation. Furthermore, we would anticipate that the association of alpha1 subunit with specific beta subunits may define developmentally distinct forms of N-type VDCC that differ in their subcellular localization and cellular function. To aid us in these studies, we have developed a battery of affinity purified antibodies which recognize the alpha1 and beta subunits. The specific aims are to (1) determine the content and diversity of VDCC alpha1 subunits expressed in crude homogenates of embryonic, neonatal and adult rat brain using quantitative western blot analysis, radioligand binding assays, and photoaffinity labelling, (2) examine the content and diversity of VDCC beta subunits in our samples using a variety of biochemical and immunological techniques and evaluate if beta and alpha1 subunits are expressed coordinately throughout neuronal development, (3) identify N-type VDCC subunits localized to distinct subcellular fractions including growth cones and synaptosomes, and (4) characterize the subunit composition of N-type VDCC complexes expressed throughout development by immunoprecipitation, immunoaffinity and biochemical purification, and immunohistochemical localization.